Isolation of nucleic acid

ABSTRACT

The present invention provides a method of isolating nucleic acid from a sample, said method comprising contacting said sample with a detergent and a solid support, whereby soluble nucleic acid in said sample is bound to the support, and separating said support with bound nucleic acid from the sample. Where the method of the invention is used to isolate DNA, it may conveniently be couple with a further step to isolate RNA from the same sample.

This Application is a continuation of, and claims priority under 35 USC§120 to U.S. application Ser. No. 11/671,426, filed Feb. 5, 2007, nowabandoned, which is a continuation of, and claims priority under 35 USC§120 to U.S. application Ser. No. 11/234,001, filed Sep. 23, 2005, nowU.S. Pat. No. 7,173,124, which is a continuation of, and claims priorityunder 35 USC §120 to U.S. application Ser. No. 08/849,686, filed Aug.21, 1997, now abandoned, which is a 35 USC §371 filing of InternationalApplication No. PCT/GB95/02893, filed Dec. 12, 1995, the disclosures ofwhich are incorporated by reference herein in their entireties.

The present invention relates to the isolation of nucleic acid, andespecially to the isolation of DNA or RNA from cells.

The isolation of DNA or RNA is an important step in many biochemical anddiagnostic procedures. For example, the separation of nucleic acids fromthe complex mixtures in which they are often found is frequentlynecessary before other studies and procedures eg. detection, cloning,sequencing, amplification, hybridisation, cDNA synthesis etc. can beundertaken; the presence of large amounts of cellular or othercontaminating material eg. proteins or carbohydrates, in such complexmixtures often impedes many of the reactions and techniques used inmolecular biology. In addition, DNA may contaminate RNA preparations andvice versa. Thus, methods for the isolation of nucleic acids fromcomplex mixtures such as cells, tissues etc. are demanded, not only fromthe preparative point of view, but also in the many methods in use todaywhich rely on the identification of DNA or RNA eg. diagnosis ofmicrobial infections, forensic science, tissue and blood typing,detection of genetic variations etc.

In RNA identifications it is important for a conclusive diagnosis to becertain that the detected sequence is derived from an RNA molecule andnot from genomic DNA contamination in the sample. For this reason,methods for the separation of RNA from DNA are important. Also, for RNAisolation rapid methods are required since RNA molecules usually arevery unstable and rapidly degraded by RNases present in cells and bodyfluids. The quality of the RNA is probably the most important factor indetermining the quality of the final results in protocols utilisingmRNA, especially for cDNA synthesis. It is important to avoid DNAcontamination of RNA preparations for a number of reasons. Firstly, DNAincreases viscosity making sample handling difficult leading to poor RNAyield and also RNA of poor quality with the liklihood of DNAcontamination. Also, DNA contamination may trap RNase enzymes and makedownstream applications such as RT-PCR worthless.

A range of methods are known for the isolation of nucleic acids, butgenerally speaking, these rely on a complex series of extraction andwashing steps and are time consuming and laborious to perform. Moreover,the use of materials such as alcohols and other organic solvents,chaotropes and proteinases is often involved, which is disadvantageoussince such materials tend to interfere with many enzymic reactions andother downstream processing applications.

Thus, classical methods for the isolation of nucleic acids from complexstarting materials such as blood or blood products or tissues involveslysis of the biological material by a detergent or chaotrope, possiblyin the presence of protein degrading enzymes, followed by severalextractions with organic solvents eg. phenol and/or chloroform, ethanolprecipitation, centrifugations and dialysis of the nucleic acids. Thepurification of RNA from DNA may involve a selective precipitation withLiCl or a selective isolation with acidic guanidinium thiocyanatecombined with phenol extractions and ethanol precipitation. Not only aresuch methods cumbersome and time consuming to perform, but therelatively large number of steps required increases the risk ofdegradation, sample loss or cross-contamination of samples where severalsamples are simultaneously processed. In the case of RNA isolation, therisk of DNA contamination is relatively high.

In purification of RNA, it is commonly desired to specifically isolatemRNA. Most mRNA purification strategies involve isolation of total RNAand fractionation of the isolated RNA. Preparation of high-quality mRNAis an important step in the analysis of gene structure and generegulation.

Most eukaryotic mRNAs have a poly(A)tail, typically about 50 to 300nucleotides long. Such mRNA is referred to as polyadenylated or poly(A)⁺mRNA. In separating this polyadenylated RNA from the non-adenylated RNAwhich accounts for 95% or more of a cell's total RNA, advantage is takenof this poly(A) tail and some type of affinity separation directedtoward the poly(A) tail is performed. The conventional technology hasinvolved purification of total RNA as a first step and selection ofpoly(A)⁺ RNA by affinity chromatography using oligo(dT)-cellulose as thesecond step. This strategy, is rather time-consuming andlabour-intensive. An alternative strategy for mRNA purification is touse oligo(dT) linked to solid supports such as microplates, latex,agarose or magnetic beads.

Over the past four years it has become increasingly popular to employ amagnetic bead assisted strategy for poly(A)⁺ RNA selection since suchbeads have proven to be favourable in mRNA manipulations. In manyapproaches, the yield and the quality of the products depends on howrapidly the mRNA can be purified from nucleases and other contaminants.By using the magnetic bead separation technology, pure, intact poly(A)⁺RNA can be obtained rapidly either from total RNA preparations or moreimportantly, directly from crude lysates of solid tissues, cell or bodyfluids. The entire procedure can be carried out in a microfuge tubewithout phenol extractions or ethanol precipitations.

One approach common in RNA purification, which may be used inconjunction with the solid phase approach is to carry out the lysis ofthe biological material and the subsequent hybridisation to oligo dT inLiCl and LiDS/SDS buffers, thereby avoiding extra steps such as phenolextraction or proteinase-K digestion. The whole direct mRNA isolationtakes approximately 15 minutes and since the mRNA is stable for morethan 30 minutes in the lysis buffer, this ensures the high quality ofthe mRNA purified. However, a disadvantage of this method is that mRNAper weight unit of tissue is affected by the amount of tissue used andabove a critical threshold of lysed cells, the yield of mRNA decreases.

Another common approach for direct mRNA purification is, as mentionedabove, to use guanidinium isothiocyanate (GTC) and sarkosyl. AGTC-buffer system is preferred by most researchers due to the ability ofthis chaotropic salt to inhibit RNases. This may also be used incombination with the magnetic bead approach. However, the viscosity ofcell lysates in 4M GTC is high and the beads are not effectivelyattracted by the magnet, resulting in an increased risk for DNAcontamination, both for beads and other solid phases, and lower yields.

More recently, other methods have been proposed which rely upon the useof a solid phase. In U.S. Pat. No. 5,234,809, for example, is describeda method where nucleic acids are bound to a solid phase in the form ofsilica particles, in the presence of a chaotropic agent such as aguanidinium salt, and thereby separated from the remainder of thesample. WO 91/12079 describes a method whereby nucleic acid is trappedon the surface of a solid phase by precipitation. Generally speaking,alcohols and salts are used as precipitants.

Although such methods speed up the nucleic acid separation process,there are disadvantages associated with the use of alcohols, chaotropes,and other similar agents. Chaotropes require to be used at highmolarity, resulting in viscous solutions which may be difficult to workwith, especially in RNA work. Amplification procedures such as PCR, andother enzyme-based reactions, are very sensitive to the inhibitory orotherwise interfering effects of alcohols and other agents. Moreover,the drying of the nucleic acid pellet which is necessary followingalcohol precipitation and the problems with dissolving nucleic acids,are also known to lead to artefacts in enzyme-based procedures such asPCR. Since such procedures are now a mainstay of molecular biology,there is a need for improved methods of nucleic acid isolation, andparticularly for methods which are quick and simple to perform and whichavoid the use of chaotropic agents or alcohol precipitation. There isalso a need for a method which allows for differentiation between RNAand DNA and permits a separate isolation of both types of nucleic acidfrom the same sample. The present invention seeks to provide suchmethods.

In particular, it has now been found that nucleic acid may be isolatedfrom a sample in a form suitable for amplification or other downstreamprocesses, by a simple and easy to perform procedure which involvestreating the sample with detergent and allowing the nucleic acid to bindto a solid support, whereupon the nucleic acid may be readily separatedfrom the sample, eg. by removal of the support. The binding of thenucleic acid is independent of its sequence.

In one aspect, the present invention thus provides a method of isolatingnucleic acid from a sample, said method comprising contacting saidsample with a detergent and a solid support, whereby soluble nucleicacid in said sample is bound to the support, and separating said supportwith bound nucleic acid from the sample.

The nucleic acid may be DNA, RNA or any naturally occurring or syntheticmodification thereof, and combinations thereof. Preferably however thenucleic acid will be DNA, which may be genomic, or, cDNA, and single ordouble stranded or in any other form.

Where the method of the invention is used to isolate DNA, it mayconveniently be coupled with a further step to isolate RNA from the samesample. The use of the method in such two-step RNA separations will bedescribed in more detail below.

The samples may be any material containing nucleic acid, including forexample foods and allied products, clinical and environmental samples.However, the sample will generally be a biological sample, which maycontain any viral or cellular material, including all prokaryotic oreukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts andorganelles. Such biological material may thus comprise all types ofmammalian and non-mammalian animal cells, plant cells, algae includingblue-green algae, fungi, bacteria, protozoa etc. Representative samplesthus include whole blood and blood-derived products such as plasma,serum and buffy coat, urine, faeces, cerebrospinal fluid or any otherbody fluids, tissues, cell cultures, cell suspensions etc.

The sample may also include relatively pure starting materials such as aPCR product, or semi-pure preparations obtained by other nucleic acidrecovery processes.

The nucleic acid-containing sample may, generally speaking, simply becontacted with the detergent, and a solid phase which may be added tothe sample prior to, simultaneously with, or subsequently to thedetergent. If necessary, this may be preceded by one or more separatesteps to disrupt structural components such as cell walls or to achievelysis. Procedures for achieving this are well known in the art. Thus,for example, although some cells eg. blood cells, may be lysed by thedetergent alone, other cells, eg. plant or fungal cells or solid animaltissues may require more vigorous treatment such as, for example,grinding in liquid nitrogen, heating in the presence of detergent,alkaline lysis in the presence of detergent. For samples in the form ofparaffin sections and such like, lysis (and melting of the paraffin) maybe effected by heating, for example using a microwave oven (Banerjee, S.K. et al., 1995, Biotechniques 18: 769-773). Also, certain more compacttissues may require enzyme treatment, for example using proteinase K toobtain sufficient release of nucleic acid. The various components aremixed and simply allowed to stand for a suitable interval of time toallow the nucleic acid to bind to the support. Conveniently, if otheragents such as enzymes eg. proteinase K are being used, they may beincluded in with the detergent. The support is then removed from thesolution by any convenient means, which will depend of course on thenature of the support, and includes all forms of withdrawing the supportaway from the sample supernatant, or vice versa, for examplecentrifugation, decanting, pipetting etc.

The conditions during this process are not critical, and it has beenfound convenient, for example, simply to mix the sample with thedetergent in the presence of a solid phase, and allow it to stand atroom temperature, for 5 to 20 minutes, before separating. As mentionedabove, the reaction time is not critical and as little as 5 minutes isoften enough. However, if convenient, longer periods may be used, eg.0.5 to 3 hours, or even overnight. Mixing can be done by any convenientmeans, including for example simple agitation by stirring or vortexing.Also, if desired, higher or lower temperatures may be used, but are notnecessary.

The detergent may be any detergent, and a vast range are known anddescribed in the literature. Thus, the detergent may be ionic, includinganionic and cationic, non-ionic or zwitterionic. The term “ionicdetergent” as used herein includes any detergent which is partly orwholly in ionic form when dissolved in water. Anionic detergents havebeen shown to work particularly well and are preferred. Suitable anionicdetergents include for example sodium dodecyl sulphate (SDS) or otheralkali metal alkylsulphate salts or similar detergents, sarkosyl, orcombinations thereof.

Conveniently, the detergent may be used in a concentration of 0.2 to 30%(w/v), eg. 0.5 to 30%, preferably 0.5 to 15%, more preferably 1 to 10%.For anionic detergents concentrations of 1.0 to 5% eg. 0.5 to 5% havebeen shown to work well.

The detergent may be supplied in simple aqueous solution, which may bealkaline or acidic, or more preferably in a buffer. Any suitable buffermay be used, including for example Tris, Bicine, Tricine, and phosphatebuffers. Conveniently, a source of monovalent cations, eg. a salt, maybe included to enhance nucleic acid capture, although this is notnecessary. Suitable salts include chloride salts, e.g. sodium chloride,lithium chloride etc. at concentrations of 0.1 to 1M, eg. 250 to 500 mM.As mentioned above, other components such as enzymes, may also beincluded.

Other optional components in the detergent composition include chelatingagents eg. EDTA, EGTA and other polyamino carboxylic acids convenientlyat concentrations of 1 to 50 mM etc., reducing agents such asdithiotreitol (DTT) or β-mercaptoethanol, at concentrations of forexample 1 to 10 mM.

Preferred detergent compositions may for example comprise:

-   -   100 mM Tris-HCl pH 7.5    -   10 mM EDTA    -   2% SDS        -   or:    -   100 mM TrisCl pH 7.5    -   10 mM EDTA    -   5% SDS    -   10 mM NaCl        -   or:    -   100 mM TrisCl pH 7.5    -   500 mM LiCl    -   10 mM EDTA    -   1% LiDS

The detergent functions in the method to lyse the nucleic acidcontaining material, eg. the cells and nuclei to release the nucleicacid. The detergent is also believed to help to disrupt the binding ofproteins, eg. DNA-binding proteins, to the nucleic acid and to reducethe problem of contaminants in the sample sticking to the solid support.

The solid support may be any of the well known supports or matriceswhich are currently widely used or proposed for immobilisation,separation etc. These may take the form of particles, sheets, gels,filters, membranes, fibres, capillaries, or microtitre strips, tubes,plates or wells etc.

Conveniently the support may be made of glass, silica, latex or apolymeric material. Preferred are materials presenting a high surfacearea for binding of the nucleic acid. Although not wishing to be boundby theoretical considerations, it is believed that the nucleic acidbinding process may be assisted by the nucleic acid “wrapping around”the support. Such supports will generally have an irregular surface andmay be for example be porous or particulate eg. particles, fibres, webs,sinters or sieves. Particulate materials eg. beads are generallypreferred due to their greater binding capacity, particularly polymericbeads.

Conveniently, a particulate solid support used according to theinvention will comprise spherical beads. The size of the beads is notcritical, but they may for example be of the order of diameter of atleast 1 and preferably at least 2 μm, and have a maximum diameter ofpreferably not more than 10 and more preferably not more than 6 μm. Forexample, beads of diameter 2.8 μm and 4.5 μm have been shown to workwell.

Monodisperse particles, that is those which are substantially uniform insize (eg. size having a diameter standard deviation of less than 5%)have the advantage that they provide very uniform reproducibility ofreaction. Monodisperse polymer particles produced by the techniquedescribed in U.S. Pat. No. 4,336,173 are especially suitable.

Non-magnetic polymer beads suitable for use in the method of theinvention are available from Dyno Particles AS (Lillestrøm, Norway) aswell as from Qiagen, Pharmacia and Serotec.

However, to aid manipulation and separation, magnetic beads arepreferred. The term “magnetic” as used herein means that the support iscapable of having a magnetic moment imparted to it when placed in amagnetic field, and thus is displaceable under the action of that field.In other words, a support comprising magnetic particles may readily beremoved by magnetic aggregation, which provides a quick, simple andefficient way of separating the particles following the nucleic acidbinding step, and is a far less rigorous method than traditionaltechniques such as centrifugation which generate shear forces which maydegrade nucleic acids.

Thus, using the method of the invention, the magnetic particles withnucleic acid attached may be removed onto a suitable surface byapplication of a magnetic field eg. using a permanent magnet. It isusually sufficient to apply a magnet to the side of the vesselcontaining the sample mixture to aggregate the particles to the wall ofthe vessel and to pour away the remainder of the sample.

Especially preferred are superparamagnetic particles for example thosedescribed by Sintef in EP-A-106873, as magnetic aggregation and clumpingof the particles during reaction can be avoided, thus ensuring uniformand nucleic acid extraction. The well-known magnetic particles sold byDynal AS (Oslo, Norway) as DYNABEADS, are particularly suited to use inthe present invention.

Functionalised coated particles for use in the present invention may beprepared by modification of the beads according to U.S. Pat. Nos.4,336,173, 4,459,378 and 4,654,267. Thus, beads, or other supports, maybe prepared having different types of functionalised surface, forexample positively charged or hydrophobic. Weakly and stronglypositively charged surfaces, weakly negatively charged surfaces, neutralsurfaces and hydrophobic surfaces eg. polyurethane-coated have beenshown to work well.

It is also possible to use solid supports which have been modified topermit the selective capture of desired cells, viruses etc. containingthe nucleic acid. Thus for example, supports carrying antibodies, orother binding proteins, specific for a desired cell type may be used.This may introduce a degree of selectivity to the isolation of thenucleic acid, since only nucleic acid from a desired target sourcewithin a complex mixture may be separated. Thus for example, such asupport may be used to separate and remove the desired cell type etc.from the sample, following which, the detergent is added to achievelysis, release of the nucleic acid, and binding to the support.

The preparation of such selective cell capture matrices is well known inthe art and described in the literature.

Likewise, the support may be provided with binding partners to assist inthe selective capture of nucleic acids. For example, complementary DNAor RNA sequences, or DNA binding proteins may be used, or viral proteinsbinding to viral nucleic acid. The attachment of such proteins to thesolid support may be achieved using techniques well known in the art.

Although not necessary, it may be convenient to introduce one or morewashing steps to the isolation method of the invention, for examplefollowing separation of the support from the sample. In the case ofmagnetic beads, this may conveniently be done before releasing the DNAfrom the beads. Any conventional washing buffers or other media may beused. Generally speaking, low to moderate ionic strength buffers arepreferred eg. 10 mM Tris-HCl at pH 8.0/10 mM NaCl. Other standardwashing media, eg. containing alcohols, may also be used, if desired.

Following the separation step, and any optional washing steps which maybe desired, the support carrying the nucleic acid may be transferred eg.resuspended or immersed into any suitable medium eg. water or low ionicstrength buffer. Depending on the support and the nature of anysubsequent processing desired, it may or may not be desirable to releasethe nucleic acid from the support.

In the case of a particulate solid support such as magnetic ornon-magnetic beads, this may in many cases be used directly, for examplein PCR or other amplifications, without eluting the nucleic acid fromthe support. Also, for many DNA detection or identification methodselution is not necessary since although the DNA may be randomly incontact with the bead surface and bound at a number of points byhydrogen bonding or ionic or other forces, there will generally besufficient lengths of DNA available for hybridisation tooligonucleotides and for amplification.

However, if desired, elution of the nucleic acid may readily be achievedusing known means, for example by heating, eg. to 65° C. for 5 to 10minutes, and following which the support may be removed from the mediumleaving the nucleic acid in solution. Such heating is automaticallyobtained in PCR by the DNA denaturation step preceding the cyclingprogram.

If it is desired to remove RNA from DNA, this may be achieved bydestroying the RNA before the DNA separation step, for example byaddition of an RNAase or an alkali such as NaOH.

Alternatively, as mentioned above, the method of the invention may beused to separate sequentially DNA and RNA from the sample. It may alsobe used to remove DNA from a sample in an RNA purification procedure.

Conveniently, the sequential separation may take place using twodifferent solid phases, for example solid supports which candifferentiate between DNA and RNA. Thus, such a method may comprisecarrying out a first step separation to isolate DNA as described above.A further solid support can then be added to the sample to capture theRNA remaining in the sample, either by using a solid support that canbind the RNA or any remaining nucleic acid, or a solid support that cancapture specific RNA molecules (eg. by carrying a complementary nucleicacid probe), or a subset of RNA molecules eg. polyadenylated RNA. Inthis way it is possible rapidly to isolate and separate DNA and RNA orsubsets of both from the same sample. This may be useful, for example bymeasuring the isolated DNA to estimate the amount of cells used for RNAextraction, which will give a reference between different samples.

However, the DNA isolation procedure of the invention may also readilybe combined, as a preliminary step, with other conventional RNApurification procedures, for example DNA isolation with detergentaccording to invention may be carried out before a selective RNAprecipitation step, for example using LiCl or before RNA separationusing GTC and sarkosyl.

In a representative procedure, the sample is lysed in the presence ofdetergent and the DNA is allowed to bind to a solid support, whereuponthe DNA may readily be separated from the sample by removal of thesupport. If desired, the DNA can rapidly and easily be further handledfor amplification or other downstream processes. The RNA may then beisolated. This can be by a solid phase based system as described above,including a repetition of the method of the invention, or byconventional techniques such as extractions, precipitations or affinitychromatography.

A particularly advantageous embodiment of the invention is to use theisolation method of the invention to remove DNA from a sample prior toisolation of RNA, such that the viscosity of the lysed sample is reducedand a specific isolation of RNA molecules is favoured which againreduces or avoids the possibility for DNA contamination of the RNA. Sucha method also has the advantage of being quick to perform.

The invention is advantageously amenable to automation, particularly ifparticles, and especially, magnetic particles are used as the support.

The various reactants and components required to perform the method ofthe invention may conveniently be supplied in kit form. Such kitsrepresent a further aspect of the invention.

At its simplest, this aspect of the invention provides a kit forisolating nucleic acid from a sample comprising a solid support and oneor more detergents.

Optionally included in such a kit may be buffers, salts, lysis agentseg. proteinases, chelating agents and reducing agents.

For isolation of RNA, the kits may further comprise means for isolatingRNA eg. a second solid support for isolating RNA, for example a supportprovided with probes for capture of RNA eg. oligo dT or probes ofcomplementary sequence to the desired target, or a chaotrope orselective precipitating agent.

The invention will now be described in more detail in the followingnon-limiting Examples with reference to the drawings in which:

FIG. 1 shows as optical density scan of DNA isolated as described inExample 1 (ordinate shows absorbance (OD), abscissa shows wavelength(nM)). Maximum absorbance (0.427) at 257.6 nM; minimum absorbance(0.292) at 236.4 nM; at a threshold of 0.100;

FIG. 2 shows gel electrophoresis of a sample of DNA isolated asdescribed in Example 1 (lane 1: isolation; lane 2: λ Hind III (molecularweight marker));

FIG. 3 shows agarose gel electrophoresis of the PCR product of Example 2(lane 1: PCR product; lane 2: λ Hind III; lane 3: negative PCR control);and

FIG. 4 shows agarose gel electrophoresis of the PCR product of Example 5(lane 1: λ Hind III; lanes 2 and 3: isolations A and B respectively;lanes 4 and 5: negative control; lane 6: λ Hind III).

FIG. 5 show the comparison between traditionally isolated DNA and DNAisolated with Dynabeads DNA DIRECT. Panel I shows the amount of genomicDNA isolated from 10 μl of whole blood with Dynabeads DNA DIRECTincluding the optional elution step (lanes 1 and 2), with Dynabeads DNADIRECT with the elution step omitted (lanes 3 and 4), and withtraditional DNA isolation (lanes 5 and 6). The molecular weight markerin lane 7 is λ HindIII. Panel II shows the integrity of DNA isolated byDynabeads DNA DIRECT. Lanes 1 and 2 show AMXY PCR from 20 ng of DNAisolated with Dynabeads DNA DIRECT from a male and female donorrespectively. Lanes 4 and 5 show AMXY PCR from 200 ng DNA isolated bytraditional methods from a female and a male donor respectively. Lane 3is the negative control.

FIG. 6 shows the reproducibility of Dynabeads DNA DIRECT. The figureshows five independent Dynabeads DNA DIRECT isolations from each of twodonors. Half of the DNA obtained from 10 μl of blood is shown in theupper part of the figure, 20% of the product from PCR reactions startedwith 10% of the isolated DNA is shown in the lower part of the figure.Molecular weight markers are λ HindIII (lanes marked M) or 100 bp ladder(lane marked L).

FIG. 7 shows the effect of different anticoagulants. The figure showsDynabeads DNA DIRECT isolations from whole blood that is notanticoagulated and from blood anticoagulated with EDTA, citrate, orheparin. The two isolations from blood with the same anticoagulant wereperformed on blood from different donors (A or B). One quarter of theDNA obtained from 10 μl of blood is shown in the upper part of thefigure, except for heparin, where half the DNA obtained from 5 μl isshown. 20% of the product from PCR reactions started with 10% of theisolated DNA is shown in the lower part of the figure, except forheparin, where 20% of the isolated DNA was used as starting material.

FIG. 8 shows the effect of sample storage conditions. Panel I showsDynabeads DNA DIRECT isolations from EDTA blood that has been usedfresh, refrigerated for 4 days, or frozen for 4 days. The two isolationsfrom blood with the same storage conditions were performed on blood fromdifferent donors. Half of the DNA obtained from 10 μl of blood is shownin the upper part of the figure, 20% of the product from PCR reactionsstarted with 10% of the isolated DNA is shown in the lower part of thefigure. Panel II shows Dynabeads DNA DIRECT isolations from citrateblood that has been used fresh or air dried and rehydrated. The twoisolations from blood with the same storage conditions were performed onblood from different donors. Half of the DNA obtained from 10.μl ofblood is shown in the upper part of the figure, 20% of the product fromPCR reactions started with 10% of the isolated DNA is shown in the lowerpart of the figure.

FIG. 9 shows Dynabeads DNA DIRECT from bone marrow and culture cells.Panel I shows Dynabeads DNA DIRECT isolations from 1, 2 and 5 μl of bonemarrow from each of two donors. A or B above the lanes denote theidentity of the donor. Half of the DNA obtained is shown in the upperpart of the figure, 20% of the product from PCR reactions started with10% of the isolated DNA is shown in the lower part of the figure. PanelII shows two Dynabeads DNA DIRECT isolations from 4×10⁵ Daudi cells. Onetenth of the DNA obtained is shown in the upper part of the figure, 20%of the product from PCR reactions started with 1 μl of a total of 120 μlisolated DNA is shown in the lower part of the figure. The molecularweight marker is λ HindIII for the genomic DNA and 100 bp ladder for thePCR products.

FIG. 10 shows Dynabeads DNA DIRECT from formalin fixed, paraffinembedded material. Lane A is 20% of the PCR product from a reactionstarted with DNA isolated by DNA DIRECT from a formalin fixed, paraffinembedded section of liver. Lane M is molecular weight marker (100 bpladder), lane B is positive control (PCR from 20 ng human DNA), and laneC is negative control (PCR from water).

FIG. 11 shows Dynabeads DNA DIRECT for mRNA purification. mRNA wasisolated from 1 million Daudi cells per sample with DynabeadsOligo(dT)₂₅ after removal of DNA with Dynabeads DNA DIRECT. Increasingamounts of DNA DIRECT Dynabeads were used to remove genomic DNA; 1 mg inlane 1 and 2; 2 mg in lane 3 and 4; 5 mg in lane 5 and 6 and 10 mg inlane 7 and 8. Lane 9 and 10 are controls where no DNA was removed beforedirect mRNA purification. The extra bands on the top of the picture showcontaminating genomic DNA in lane 9 and 10. The two strong bands in alllanes represent ribsomal RNA.

FIG. 12 shows the results of DNA isolation and PCR amplification from(A) bacteria, (B) fungi, (C) algae and (D) plants. For all samples, DNAwas isolated with 200 μl DNA DIRECT (one sample test) and 20% of theisolated DNA and 10% of the PCR products were analysed by agaroseelectrophoresis. For bacteria, 2.5% of the isolated DNA was used per PCRreaction, for the other samples 5% was used. 16S rRNA regions wereamplified from bacterial genomic DNA and from algae chloroplast DNA.From fungi- and algae genomic DNA, 18S rDNA were amplified.Amplification of the group I intron chloroplast trnL and parts of thegenomic B15C gene are shown for plants. The negative controls are PCR onsamples without DNA prepared in the same way as the other reactions.

EXAMPLE 1 DNA Isolation from Cell Culture

4×10⁶ HL60 cells were washed twice in PBS and pelleted. The pellet wasdissolved in 10 μl PBS, and 1 mg of Dynabeads® M-280* obtainable byautoclaving a suspension of Dynabeads® M-280 tosylactivated (availablefrom DYNAL A/S, Oslo, Norway) in water) resuspended in 0.1 ml lysisbuffer [5% SDS/10 mM TrisCl pH 8.0/1 mM EDTA] was added. This wasfollowed immediately by the addition of 1 ml lysisbuffer, and thesuspension was incubated for 5 minutes at room temperature, after whichthe Dynabeads®, with bound DNA was attracted to a magnet and the liquidphase removed. The solid phase was then washed twice with 1 ml washingbuffer [50 mM NaCl/10 mM TrisCl pH 8.0/1 mM EDTA]. Finally, the beads,with bound DNA, were resuspended in 0.1 ml water, and incubated for 5minutes at 65° C. The beads were attracted to a magnet, and the liquidphase withdrawn. The liquid phase was then analyzed for its DNA content.Results from an optical density scan (FIG. 1) are in accordance withpure DNA. The OD₂₆₀/OD₂₈₀ ratio is 1.72; pure DNA in water or TE has aratio of 1.7-1.9. With pure DNA, the concentration can be determinedfrom the OD₂₆₀ of the solution. A 50 μg/ml solution has OD₂₆₀=1.0. Fromthe OD₂₆₀ measurement (Table 1) of 0.436 (0.1 ml total volume, 10 mmlightpath), the yield can be calculated to 2.18 μg DNA, 82% of the 2.67μg that was the estimated DNA content of the starting material. Gelelectrophoresis of a sample of the isolated DNA (FIG. 2) shows that mostof it is in a high molecular weight form (>20 kb).

TABLE 1 PERKIN-ELMER LAMBDA BIO UV/VIS SPECTROMETER APPLICATION NO. 3:RATIO 260/280 NM WAVELENGTH SAMPLE CYCLE AUTOZERO DATA UNIT 004 15:50260.0 nm 0.436 ABS 15:56 280.0 nm 0.253 ABS RATIO 1.723 RAT

EXAMPLE 2 Isolation of DNA from Whole Blood and Enzymatic Applicationwithout Elution

5 μl whole blood (EDTA blood) was lysed in 50 μl 5% SDS and 50 μgDynabeads® M-280* in 5 μl of PBS was added. The lysate was incubated for1 minute at room temperature before 0.5 ml TrisCl pH 7.5 was added. Thelysate was then incubated for 1 minute further at room temperaturebefore the beads with bound DNA were attracted to a magnet and theliquid phase removed. The beads were then washed once with 0.5 ml 10 mMTrisCl pH 7.5, before the beads with bound DNA was resuspended in 40 μlTE (10 mM TrisCl pH 8.0/1 mM EDTA). 4 μl of the isolation was used instarting material for PCR (GAPDH PCR as described in Example 7). The PCRreaction gave large amounts of product, as visualised on agarose gelelectrophoresis (FIG. 3). 10 μl of a 50 μl PCR reaction was loaded onthe gel.

EXAMPLE 3

Example 1 was repeated using the following combination of lysisbuffersand washing buffers, and the following results were obtained:

(where +++ indicates very good DNA isolations

Lysis buffer Washing buffer Result 2% SDS 50 mM NaCl/1 × TE +++ 2% SDS/1× TE 50 mM NaCl/1 × TE +++ 2% SDS/1 × TE/10 mM NaCl 50 mM NaCl/1 × TE+++ 5% SDS 50 mM NaCl/1 × TE +++ 5% SDS/1 × TE 50 mM NaCl/1 × TE +++ 5%SDS/1 × TE/10 mM NaCl 50 mM NaCl/1 × TE +++ 1% LiDS/10 × TE/0.5 M LiCl50 mM NaCl/1 × TE +++ 1% LiDS/10 × TE/0.5 M LiCl 150 mM LiCl/1 × TE  +++5% LiDS 150 mM LiCl/1 × TE  +++ 5% SDS 150 mM LiCl/1 × TE  +++ 1%Sarcosyl 150 mM LiCl/1 × TE  +++1×TE is 10 mM TrisCl pH 8.0/1 mM EDTA, 10×TE is 100 mM Tris Cl pH 8.0/10mM EDTA

EXAMPLE 4

Following the procedure of Example 1, similar results may be achievedusing Dynabeads® M-450 uncoated (Dynal A/S, Oslo, Norway)

EXAMPLE 5 Isolation of DNA from CD2 Positive Cells Obtained from Bloodwith Immunomagnetic Separation

This experiment consisted of two identical isolations. 50 μl blood wasmixed with 50 μl PBS [150 mM NaCl/10 mM NaH₂PO₄/Na₂HPO₄, pH 7.4] and 10μl (4×10⁶ beads) Dynabeads® M-450 Pan-T (CD2) (available from Dynal AS,Oslo, Norway). The mixture was then incubated for 30 minutes at roomtemperature with gentle tilting and rotation. The cell/beads complex wasattracted to a magnet and the fluid withdrawn. The cell/beads complexwas then washed four times in 200 μl PBS, before 200 μg Dynabeads®M-280* (as above) and 200 μl lysisbuffer [100 mM Tris-HCl, pH 8.0/500 mMLiCl/10 mM EDTA, pH 8.01/1% LiDS] was added. The mixture was incubatedfor 5 minutes at room temperature, before the DNA/beads complex wasattracted to a magnet, and the supernatant withdrawn. The DNA/beadscomplex was washed twice with 200 μl washing buffer [10 mM Tris-Hcl, pH8.0/150 mM LiCl/1 mM EDTA, pH 8.0] and resuspended in 50 μl water. After5 minutes at 65° C., the beads were attracted to a magnet and thesupernatant transferred to a new tube. 5 μl of the supernatant was usedas template for polymerase chain reaction (GAPDH PCR as described inExample 7), which gave large amounts of product, as visualised onagarose gel electrophoresis (FIG. 4).

EXAMPLE 6 Comparison of Yield and Integrity Between DNA Isolated by aTraditional Method and the Present Method

Using a traditional method based on phenol extraction and ethanolprecipitation, genomic DNA was isolated from 5 ml of EDTA anticoagulatedblood. Four isolations from 10 μl of the same blood sample wereperformed using Dynabeads DNA DIRECT (kit, commercially available fromDynal AS, Oslo, Norway, containing beads equivalent to Dynabeads® M-280*as described in Example 1). The DNA from two of the isolations waseluted for 5 minutes at 65° C., while the DNA from the other twoisolations was left in the presence of the Dynabeads. All the DNA fromthe four Dynabeads DNA DIRECT isolations was loaded onto an agarose gel,as was 0.2% of traditionally isolated DNA. The fraction of thetraditionally isolated DNA loaded corresponds to the yield from 10 μl ofblood (0.2% of 5 ml).

Traditional DNA isolation was performed according to the method of Johnand coworkers (John, S. W. M., G. Weitzner, R. Rosen and C. R. Scriver.1991. A Rapid Procedure for Extracting Genomic DNA from Leukocytes.Nucl. Acid. Res. 19(2):408).

With Dynabeads DNA DIRECT, lysis of the blood was obtained by mixing 200μl (one sample test) of Dynabeads DNA DIRECT with 10 μl of blood in a1.5 ml microcentrifuge tube (200 μg uncoated Dynabeads in Lysis/bindingbuffer). Lysates were then left on the bench at room temperature for 5minutes to allow adsorption of genomic DNA to the Dynabeads.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E (MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 10 μl of TE pH 8.0(provided in the kit).

When elution was performed, it consisted of heating the suspension to65° C. for five minutes and then attracting the beads to a magnet. TheDNA-containing aqueous phase was then withdrawn and used for theexperiments.

The DNA was visualised on ethidium bromide stained 1.5% agarose gels.Electrophoresis was performed in 1×TAE buffer, and the results weredocumented with a DS34 Polaroid camera and Polaroid 667 film.

The result of this experiment is shown in panel I of FIG. 5. The yieldper μl blood is similar with the two methods (lane 1-4 vs lane 5-6), andvery little DNA is lost during the elution step (lane 1-2 vs lane 3-4).The molecular weight of the DNA from both methods is more than 20 kb, asit runs slower than the 23.13 kb band of the λ HindIII molecular weightmarker.

DNA DIRECT was used to isolate DNA from ACD blood from two differentdonors, one male and one female. From each of the isolations, 10% wasused as starting material for PCR amplification of an amplicon in theX-Y homologous amelogenin (AMXY) gene (Akane, A., K, Matsubara, H.Nakamura, S. Takahashi and K. Kimura. 1994. Purification of HighlyDegraded DNA by Gel Filtration for PCR. BioTechniques 16 (2):235-238),as was 200 ng from each of two traditional DNA isolations.

DNA DIRECT and traditional DNA isolation was performed as described inthe first part of this example.

All PCR reactions were performed in a 50 μl reaction volume, 10×PCRbuffer (Perkin Elmer) was added to a final concentration of 1×, dNTPs(Pharmacia) were added to a final concentration of 0.2 mM, and 1 unit ofamplitaq (Perkin Elmer) was used per reaction. 5 pmol each of primersAMXY-1F (5′-CTGATGGTTGGCCTCAAGCCT-GTG-3′) and AMXY-4R(5′-TTCATTGTAAGAGCAAAGCAAACA-3′) were added per reaction. PCR wasperformed on a Perkin Elmer GeneAmp PCR System 9600. PCR conditions forthe AMXY amplicon were 4 min at 94° C., 38×[30 sec at 94° C., 30 sec at55° C., 1 min at 72° C.], 10 min at 72° C.

10 μl of the 50 μl PCR reactions were visualised on ethidium bromidestained 1.5 agarose gels. Electrophoresis was performed in 1×TAE buffer,and the results were documented with a DS34 Polaroid camera and Polaroid667 film.

The results of this experiment are shown in panel II of FIG. 5. The X-Yhomologous amelogenin gene is known to be sensitive to DNA degradation(Akane et al 1994, supra). With increasing degradation, the 908 bp longX band gets progressively weaker as compared to the 719 bp long Y band.From panel II of FIG. 5 it is apparent that the relative strength of theX and Y bands is comparable for DNA isolated with Dynabeads DNA DIRECTand the traditional method, indicating that the degree of degradation isthe same with the two methods. The PCR reactions from traditionallyisolated DNA gives somewhat more product than does the reactions fromDNA DIRECT isolated DNA. The reason for this is that ten times moretemplate is used in the PCR reactions from traditionally isolated DNAthan in the PCR reactions from DNA DIRECT isolated DNA.

Lysis/binding buffer: 0.5 M LiCl 1% LiDS 0.1 M TrisCl pH 7.5 10 mM EDTA5 mM dithiothreitol (DTT) Washing buffer: 0.15 M LiCl 10 mM Tris-HCl pH8.0 1 mM EDTA

EXAMPLE 7 Five Independent DNA Isolations from Blood Samples from Eachof Two Donors

For this experiment, we used Dynabeads DNA DIRECT kit, which iscommercially available from Dynal AS, Oslo, Norway. Buffer compositionsare as described in example 6.

Five DNA isolations were performed from each of two citrate treatedblood samples of relatively low white blood cell counts (sample A:3.6×10⁶ cells/ml, sample B: 2.6×10⁶ cells/ml).

Lysis of the blood was obtained by mixing 200 μl (one sample test) ofDynabeads DNA DIRECT with blood in a 1.5 ml microcentrifuge tube.Lysates were then left on the bench at room temperature for 5 minutes toallow adsorption of genomic DNA to the Dynabeads.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E (MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 40 μl of TE pH 8.0(provided in the kit). This resuspension was used for PCR and gelelectrophoresis without any elution.

From each isolation, 10% was used as starting material for PCRamplification of an amplicon in the glyceraldehyde phosphatedehydrogenase (GAPDH) gene. All PCR reactions were performed in a 50 μlreaction volume, 10×PCR buffer (Perkin Elmer) was added to a finalconcentration of 1×, dNTPs (Pharmacia) were added to a finalconcentration of 0.2 mM, and 1 unit of amplitaq (Perkin Elmer) was usedper reaction. 5 pmol each of primers GAPDH-Forward(5′-ACAGTCCATGCCATCACTGCC-3′) and GAPDH-Reverse(5′-GCCTGCTTCACCACCTTCTTG-3′) were added per reaction. PCR was performedon a Perkin Elmer GeneAmp PCR System 9600. PCR conditions for the GAPDHamplicon were 4 min at 94° C., 34×[30 sec at 94° C., 30 sec at 61° C., 1min at 72° C.], 10 min at 72° C.

Both genomic DNA and PCR products were visualised on ethidium bromidestained 1.5% agarose gels. 10 μl of the 50 μl reaction was loaded on toan agarose gel, as was 50% of the isolated genomic DNA. Electrophoresiswas performed in 1×TAE buffer, and the results were documented with aDS34 Polaroid camera and Polaroid 667 film.

The results of this experiment are shown in FIG. 6. No significantvariation between the different isolations can be observed. Similarresults were obtained with other coagulants as well as from donors withhigher white blood cell counts.

EXAMPLE 8 DNA Isolation from Blood with Different Anticoagulants

Dynabeads DNA DIRECT (kit, commercially available from Dynal AS, Oslo,Norway) was used to isolate DNA from untreated whole blood as well asblood anticoagulated with EDTA, Citrate or Heparin. From each type ofstarting material, two separate isolations were performed, with bloodfrom different donors. The buffer components in the kit are as describedin example 6.

Lysis of the DNA containing cells from blood was obtained by mixing 200μl (one sample test) of Dynabeads DNA DIRECT with 5 μl Heparin blood or10 μl of other blood samples in a 1.5 ml microcentrifuge tube. Lysateswere then left on the bench at room temperature for 5 minutes to allowadsorption of genomic DNA to the Dynabeads.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E (MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 20-40 μl of TE pH8.0 (provided in the kit). We used 40 μl as the standard volume, but 20μl if the starting material was Heparin blood.

From each isolation, 10% (20% from the Heparin samples) was used asstarting material for PCR amplification of an amplicon in theglyceraldehyde phosphate dehydrogenase (GAPDH) gene. PCR was performeddirectly on the suspension in TE, with the Dynabeads present. All PCRreactions were performed in a 50 μl reaction volume, 10×PCR buffer(Perkin Elmer) was added to a final concentration of 1×, dNTPs(Pharmacia) were added to a final concentration of 0.2 mM, and 1 unit ofamplitaq (Perkin Elmer) was used per reaction. 5 pmol each of primersGAPDH-Forward (5′-ACAGTCCATGCCATCACTGCC-3′) and GAPDH-Reverse(5′-GCCTGCTTCACCACCTTCTTG-3′) were added per reaction. PCR was performedon a Perkin Elmer GeneAmp PCR System 9600. PCR conditions for the GAPDHamplicon were 4 min at 94° C., 34×[30 sec at 94° C., 30 sec at 61° C., 1min at 72° C.], 10 min at 72° C.

10 μl of the 50 μl reaction was loaded on to an agarose gel, as was 25%(50% from the Heparin samples) of the isolated genomic DNA. Both genomicDNA and PCR products were visualised on ethidium bromide stained 1.5%agarose gels. Electrophoresis was performed in 1×TAE buffer, and theresults were documented with a DS34 Polaroid camera and Polaroid 667film.

The results of this experiment are shown in FIG. 7. As the isolationsfrom Heparinized samples were from only 5 μl of blood, using 20% of theDNA from these isolations as starting material for PCR is comparable tousing 10% from the other isolations, that are all from 10 μl blood. Whenthis is taken into consideration, it is apparent that the type ofanticoagulant used does not significantly affect the result.

In the experiment just described, Lithium Heparin was used. In thissystem, similar results are obtained with Lithium and Sodium Heparin,even though Lithium Heparin has been shown to have inhibitory effects inother systems (Panaccio, M., M. Georgesz and A. M. Lew. 1993. FoLT PCR:A Simple PCR Protocol for Amplifying DNA Directly from Whole Blood.BioTechniques 14(3): 238-243). DNA DIRECT also performs well on bloodanticoagulated with ACD (panel II of FIG. 5) or CPD (data not shown).

EXAMPLE 9 Isolation of DNA from Blood Samples Stored Under DifferentConditions

Dynabeads DNA DIRECT (kit, commercially available from Dynal AS, Oslo,Norway) was used to isolate DNA from EDTA blood from two differentdonors. What was remaining of the blood samples were then divided intotwo, one part that was stored at +4° C. and one that was stored at −20°C. After 4 days, the frozen samples were thawed, and DNA was isolatedfrom both the frozen samples and the samples that had been kept at +4°C. The buffer components of the kit are as described in example 6.

Lysis of the blood was obtained by mixing 200 μl (one sample test) ofDynabeads DNA DIRECT with blood in a 1.5 ml microcentrifuge tube.Lysates were then left on the bench at room temperature for 5 minutes toallow adsorption of genomic DNA to the Dynabeads.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E (MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 40 μl of TE pH 8.0(provided in the kit). Both PCR and agarose gel electrophoresis wasperformed directly on the suspension in TE, with the Dynabeads present.

From each of the 6 isolations (fresh, refrigerated, and frozen), 10% wasused as starting material for PCR amplification of the GAPDH amplicon asdescribed in example 8. Both genomic DNA and PCR products werevisualised on ethidium bromide stained 1.5% agarose gels. 10 μl of the50 μl reaction was loaded on the gel, as was 50% of the isolated genomicDNA. Electrophoresis was performed in 1×TAE buffer, and the results weredocumented with a DS34 Polaroid camera and Polaroid 667 film. Theresults of this experiment are shown in panel I of FIG. 8. No adverseeffect of 4 days storage at +4 or −20° C. was observed in this system.Using Dynabeads DNA DIRECT as described earlier in this example, DNA wasisolated from two citrate treated blood samples, and from the same twosamples 10 μl was spotted on a plastic surface and allowed to air dry atroom temperature. The dried blood spots were transferred to 1.5 mltubes, 40 μl PBS was added, and the tubes were left at room temperaturewith gentle agitation for 90 min, before DNA was isolated with DynabeadsDNA DIRECT. From each of the 4 isolations (fresh and dried), 10% wasused as starting material for PCR amplification of the GAPDH amplicon asdescribed in example 8. Both genomic DNA and PCR products werevisualised on ethidium bromide stained 1.5% agarose gels. 10 μl of the50 μl reaction was loaded on the gel, as was 50% of the isolated genomicDNA. Electrophoresis was performed in 1×TAE buffer, and the results weredocumented with a DS34 Polaroid camera and Polaroid 667 film. Theresults of this experiment are shown in panel II of FIG. 8. The yieldfrom dried blood is good and the isolated DNA is suitable for PCR.

EXAMPLE 10 DNA Isolation from Bone Marrow and Culture Cells

DNA Isolations from Bone Marrow

1, 2, and 5 μl of heparinized bone marrow from each of two healthydonors were used as starting material for DNA isolation with DNA DIRECT.The buffer components are as described in example 6. Lysis of the bonemarrow was obtained by mixing 200 μl (one sample test) of Dynabeads DNADIRECT with 1-5 μl of heparinized bone marrow in a 1.5 mlmicrocentrifuge tube. Lysates were then left on the bench at roomtemperature for 5 minutes to allow adsorption of genomic DNA to theDynabeads.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E(MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 40 μl of TE pH 8.0(provided in the kit). Both PCR and agarose gel electrophoresis wasperformed directly on the suspension in TE, with the Dynabeads present.

From each of the 6 isolations, 10% was used as starting material for PCRamplification of the GAPDH amplicon as described in example 8.

Both genomic DNA and PCR products were visualised on ethidium bromidestained 1.5% agarose gels. 10 μl of the 50 μl reaction was loaded on toan agarose gel, as was 50% of the isolated genomic DNA. Electrophoresiswas performed in 1×TAE buffer, and the results were documented with aDS34 Polaroid camera and Polaroid 667 film.

The results of this experiment are shown in panel I of FIG. 9. It isapparent from panel I of FIG. 9 that the yield per volume startingmaterial is higher from bone marrow than from blood (FIGS. 5-8). This isto be expected, since the concentration of DNA containing cells is muchhigher in bone marrow than in blood. 5 μl of bone marrow is close to theupper limit of what can be handled by 1 sample test of DNA DIRECT. Agood correlation between sample size and DNA yield is observed in the 1to 5 μl sample size interval, but even the yield from 1 μl is sufficientfor at least 10 PCR reactions.

DNA Isolation from Cultured Cells

Two samples of 4×10⁵ Daudi cells were used as starting material for DNAisolation with DNA DIRECT. DNA isolation from 4×10⁵ cultured cells (cellline Daudi) was performed as described above, except that 1 ml (fivesample tests) of Dynabeads DNA DIRECT was used. Accordingly, the washingsteps were performed in 1 ml washing buffer. The DNA/Dynabeads complexwas resuspended in 120 μl TE, and as for bone marrow, no elution stepwas performed after the resuspension.

From each of the isolations, 1 μl of a total of 120 μl was used asstarting material for PCR amplification of the GAPDH amplicon, asdescribed in Example 8.

Both genomic DNA and PCR products were visualised on ethidium bromidestained 1.5% agarose gels. 10 μl of the 50 μl reaction was loaded on toan agarose gel, as was 10% of the isolated genomic DNA. Electrophoresiswas performed in 1×TAE buffer, and the results were documented with aDS34 Polaroid camera and Polaroid 667 film.

The results of this experiment are shown in panel II of FIG. 9,demonstrating that at least 120 PCR reactions may be run from anisolation of this scale.

EXAMPLE 11 Isolation of DNA from a Formalin Fixed, Paraffin EmbeddedSection of Liver

Dynabeads DNA DIRECT (kit, commercially available from Dynal AS, Oslo,Norway) was used to isolate DNA from a formalin fixed, paraffin embeddedsection of liver. The buffer components of the kit and the beadconcentration are as described in example 6.

Excess paraffin was removed from the edges of the sample with a scalpelblade. Lysis of the sample was obtained by adding 200 μl (one sampletest) of Dynabeads DNA DIRECT to the sample in a 1.5 ml microcentrifugetube. Lysates were then left on the bench at room temperature for 5minutes to allow adsorption of genomic DNA to the Dynabeads. The lysate,containing DNA and Dynabeads was transferred to a fresh tube, leavingcell debris and paraffin behind.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E (MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 10 μl of sterilewater. This suspension, with the Dynabeads present, was used as startingmaterial for PCR amplification of the GAPDH amplicon as described inexample 8. The PCR product was visualised on an ethidium bromide stained1.5% agarose gel. 10 μl of the 50 μl reaction was loaded on the gel.Electrophoresis was performed in 1×TAE buffer, and the results weredocumented with a DS34 Polaroid camera and Polaroid 667 film. The resultof this experiment is shown in FIG. 10. PCR amplifiable material hasclearly been obtained from the formalin fixed, paraffin embedded sectionof liver.

EXAMPLE 12 Removal of Genomic DNA with DNA DIRECT Prior to mRNAIsolation

mRNA was isolated from 1 million Daudi cells per sample. The cells werelysed in 0.75 ml Lysis/binding buffer with DNA DIRECT Dynabeads presentin the buffer. The samples were incubated for 5 minutes and theDNA-Dynabead complexes were collected by applying a Dynal MPC-E magnetfor 2 minutes. Different amounts of DNA DIRECT beads were used to removegenomic DNA; 1, 2, 5 and 10 mg per sample (FIG. 11).

The lysate from each sample was transferred to new tubes with 1 mgDynabeads Oligo(dT)₂₅ according to standard procedure (Dynals mRNADIRECT kit protocol). The Dynabeads were mixed with the lysate tocapture the polyadenylated mRNA by hybridisation for 5 minutes at roomtemperature. The mRNA-Dynabead complexes were collected with the MPC-Emagnet by placing the tubes in the magnetic stand for 2 minutes. Thesolution was removed and discarded. Washing solution with LiDS (0.75 ml)was added and the beads were washed thoroughly by pipetting up and down.The mRNA-Dynabead complexes were collected with the magnet, and thewashing procedure was repeated once with washing buffer with LiDS andtwice with washing buffer without detergent. Finally, the purified mRNAwas eluted from the Dynabeads in 20 μl 5 mM Tris-HCl pH 7.5 buffer, byincubation at 65° C. for 2 minutes. The eluates were analysed bynon-denaturing gel electrophoresis in a 1.0% agarose gel with ethidiumbromide. FIG. 11 shows the results from this experiment.

The EtBr-staining reveals both double-stranded DNA and rRNA due tosecondary structure. The two ribosomal RNA bands are clearly visible inall lanes. In Lane 9-10 in FIG. 11, some genomic DNA is present ascontamination after the mRNA purification procedure. In the recommendedmRNA procedure from DYNAL, a DNA-shearing step is introduced after celllysis, to reduce the possibility of DNA contamination. By using DNAremoval with DNA DIRECT beads this shearing step is not necessary.

Lysis/binding buffer: 0.5 M LiCl 1% LiDS 0.1 M TrisCl pH 7.5 10 mM EDTA5 mM dithiothreitol (DTT) Washing buffer with LiDS: 0.15 M LiCl 0.1%LiDS 10 mM Tris-HCl pH 8.0 1 mM EDTA Washing buffer: 0.15 M LiCl 10 mMTris-HCl pH 8.0 1 mM EDTA

EXAMPLE 13 Universal Method for DNA Isolation PCR-ready DNA fromBacteria, Fungi, Algae, Plants and Vertebrates

E. coli and Baceillus cereus were grown overnight at 37° C. in LBmedium, Agrobacterium tumefaciens was grown overnight in YEB medium forabout 40 hours at 28° C. (Sambrook, J. et al., 1989, Molecular Cloning:A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory, NY.).Cyanobacteria and Prochlorthrix were grown in NIVA medium for 14 days at18° C. using an illumination of 20 micro Einstein (Norwegian Instituteof Water Research, 1991, Culture collection of algae). 20-200 millionbacteria or 450,000 cyanobacteria were used per DNA isolation.

Agar plates containing 2% malt extract was used for mycelia growth andincubated for 14 days at room temperature. Mycelia was isolated byscraping the surface of the agar plates with a spatula. Fungifruitbodies were obtained from natural populations. In the range of 1-3mg air dried and 3-20 mg fresh fungi fruitbodies were used per DNAisolation.

Bakers yeast Sacchaomyces cerevisiae was obtained from a commercialsupplier. Algae were cultured under illumination in IMR-medium for 7days (Eppley, R et al., 1967, Exp. Mar. Biol. Ecol. 1, 191-208). Freshleaves from Arabidopsis thaliana and barley (Hordeum vulgare) werepicked from young plants (3 weeks old). Epithelia were obtained fromperch (Perca fluvatilis) fins. About 1 mg wet weight yeast, 30-100 mgyoung plant leaves and 100-400 mg perch were used per DNA isolation.

Multicellular tissues with rigid cell walls were mechanically broken toincrease DNA yield. Fungi fruitbodies were ground with foreceps forabout 2 minutes. Plant leaves were homogenised for 2 minutes in liquidnitrogen with a pestle (Kontes Scientific Instruments, Vineland, N.J.,USA). For all other samples no mechanical work was required for cellbreakage.

DNA Isolation

DNA isolations were performed using Dynabeads DNA DIRECT (kit,commercially available from Dynal AS, Oslo, Norway). Lysis of the cellsand organisms were obtained by mixing 200 μl of Dynabeads DNA DIRECT(200 μg uncoated Dynabeads in Lysis/binding buffer) with the sample in a1.5 ml microcentrifuge tube. Lysates were then left on the bench at roomtemperature for 5 to 15 minutes to allow adsorption of genomic DNA tothe Dynabeads. For some bacteria and for plants, incubation at 65° C.for 15 minutes was used to improve lysis before the adsorption step.

The DNA/Dynabeads complex was attracted to a magnet (Dynal's MagneticParticle Collector E (MPC-E)), and the lysate was aspirated anddiscarded.

The complex was then washed twice in washing buffer (one of the kitcomponents) by attracting it to a Dynal MPC and discarding thesupernatant. Finally, the complex was resuspended in 40 μl of TE pH 8.0(provided in the kit) by vigorous pipetting. Elution was performed byheating the suspension to 65° C. for five minutes and then attractingthe beads to a magnet. The DNA-containing aqueous phase was thenwithdrawn and used for the experiments.

The DNA was visualised on ethidium bromide stained 1.5% agarose gels.Electrophoresis was performed in 1×TAE buffer, and the results weredocumented with a DS34 Polaroid camera and Polaroid 667 film.

Lysis/binding buffer: 0.5 M LiCI 1% LiDS 0.1 M TrisCl pH 7.5 10 mM EDTA5 mM dithiothreitol (DTT) Washing buffer: 0.15 M LiCl 10 mM Tris-HCl pH8.0 1 mm EDTA

For an evaluation of the yield from the DNA DIRECT isolation protocol,standard phenol/chloroform based methods were used. Algae, vertebrateand bacterial DNA were isolated with the protocol described by Sambrook,J. et al., 1989, supra. Cyanobacteria were homogenized with alumina typeA-5 (Sigma Chemicals Co., St. Louis, USA) before isolation to ensurecomplete lysis. Plant and fungi DNA were isolated with the protocoldescribed by Scot, O. R. and Bendich, A. J., 1994, in “Plant MolecularBiology Manual”, page D1: 1-8, Kluwer Academic Publisher, Belgium.

PCR Amplifications

For each sample type the reproducibility was tested by using separateDNA isolations, serial DNA dilutions and multiple PCR assays. DNAisolation reagents and PCR reagents were controlled for absence ofcontamination in each separate experiment. All PCR reactions wereperformed in a 50 μl reaction volume containing; 15 pmoles primers, 200μM dNTP, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl₂, 50 mM KCl, 0.1% TritonX-100, 1 Unit DynaZyme thermostable polymerase (Finnzymes Oy, Finland)and 0.1-5 μl of isolated DNA. PCR was performed on a Perkin ElmerGeneAmp PCR System 9600.

Amplicons and Oligonucleotide Primers

All PCR reactions were started with a DNA denaturation step at 94-97° C.for 3 to 5 minutes and ended with an extension step at 72° C. for 5minutes.

Bacteria and Algae:

The amplicon was a 16S rRNA region corresponding to E. coli base 334 to939 according to IUD numbering from bacteria and algae chloroplasts(Brosius, J., et al., 1978, Proc. Natl. Acad. Sci., USA, 57, 4801-4805).

Primers: CC 5′-TGTAAAACGACGGCCAGTCCAGACTCCTACGGGAGGCAGC-3′ CD5′-CTTGTGCGGGCCCCCGTCAATTC-3′

Primer CC has a 5′ end complementary to −21 M13 universal primer, makingit suitable for direct DNA sequencing. Amplification: 30 cycles of 96°C. for 15 seconds and 70° C. for 2 minutes.

Algae: An 18S rRNA region was amplified with the primers A and Bdescribed by Medlin et al., 1990, Gene, 71, 491-499.

Amplification: 35 cycles of 94° C. for 30 seconds, 50° C. for 1 minuteand 72° C. for 1 minute.

Fungi: A 18S rRNA region (ca. 600 bp.) was amplified with the primersNS3 and NS4 as described by White et al., 1990. In “PCR Products, aGuide to Methods and Applications” by Innis, M. A. et al., page 315-322,Academic Press, New York.

Amplification: 5 cycles of 94° C. for 30 seconds, 53° C. for 30 secondsand 72° C. for 1 minute; followed by 25 cycles of 94° C. for 30 seconds,50° C. for 30 seconds increasing with 15 seconds for each cycle, and 72°C. for 1 minute.

Plants: The tRNL group I intron in chloroplasts were amplified with theprimers C and D described by Fangan et al., 1994, BioTechniques 16,484-494.

Amplification: 30 cycles of 94° C. for 30 seconds, 55° C. for 30 secondsand 72° C. for 1 minute.

A part of the Arabidopsis thaliana gene BI5C (800 bp) was amplified withthe primers

5′-CGGGATCCCTAGGAGACACGGTGCCG-3′ and 5′-GGAATTCGATCGGCGGTCTTGAAAC -3′

Amplification: 35 cycles of 94° C. for 30 seconds, 59° C. for 30 secondsand 72° for 1 minute.

A part of the Barley gene Bl5C (800 bp) was amplified with the primers5′-CGGATCCCGTCATCCTCTTCTCGCACCCC-3′ and5′-GGAATTCCCTTCTTGGAGGGCAGGTCGGCG-3′.

Amplification: 35 cycles of 94° C. for 30 seconds, 60° C. for 30 secondsand 72° C. for 1 minute.

Perch: Mitochondrial D-loop fragment (800-900 bp) was amplified with theprimers HV2 described by Hoelzel et al., 1991, Mol. Biol. Evol., 8,475-493, and the primer 5′-GGTGACTTGCATGTGTAAGTTCA-3′.

Amplification: 30 cycles of 96° C. for 1 minute, 52° C. for 2 minutesand 72° C. for 2 minutes.

The amplified fragments were visualised on ethidium bromide stained 1.5%agarose gels. Electrophoresis was performed in 1×TAE buffer, and theresults were documented with a DS34 Polaroid camera and Polaroid 667film.

The results of the experiments are shown in FIG. 12 and Table 2.

Bacteria: The standard protocol gave DNA yields in the range of 100-1000ng for the bacteria tested (FIG. 12A). For some Cyanobacteria there wasa substantial increase in DNA yield (from 500 ng to more than 1 mg) byimproving the lysis with an extra initial incubation step at 65° C. for15 minutes. In all cases, good amplifications were obtained by using0.25% of the isolated DNA.

Fungi: The highest DNA yield was obtained from dried fruit-bodies(300-500 ng) compared with fresh fruit bodies (100-200 ng) (FIG. 12B).Mycelia gave low DNA recovery probably due to low number of cells persample. However, in most cases 5% of the isolated DNA was enough to givenice PCR products (Table 2, FIG. 12B). For fruit bodies, 0.5-5% of DNAwas used for each PCR.

Algae: All algae tested gave DNA yield in the range of 200-400 ng usingthe standard protocol and 1 DNA DIRECT sample test (Table 2, FIG. 12C).Nice PCR results were obtained both for amplification of genomic DNA andchloroplast DNA by using 5% of the isolated DNA per PCR reaction.

Plants: To obtain good DNA yield from plant leaves homogenization inliquid nitrogen was necessary. An extra initial incubation step at 65°C. for 15 minutes also improved the results (FIG. 12D). Nice PCR resultswere obtained both for amplification of genomic DNA and chloroplast DNA,when 5% of the isolated DNA was used per PCR reaction.

Fish: DNA yield of 300-500 ng was routinely obtained from fishepithelia, using the standard protocol and one sample test (Table 2).Mitochondrial DNA was nicely amplified using 5% of the isolated DNA.

PCR products from all species tested could easily be directly sequencedby solid-phase sequencing (Hultman et al., 1989, Nucleic Acids Res., 17,4937-4946).

TABLE 2 DNA isolation and PCI amplIfIcation from different organisms

^(a) A. tumerfaciens = Aghrobacterium tumefaciens, P. bollandica =Prochlorothrix hollandica. ^(b)d. fruitb. = dried fruitbodies, f.mycelia = fresh mycelia, ep. = epilthelium. ^(c)Approximate DNA yieldsrelative to standart phenol/chloroform isolations; +++: >80%, ++: >10%,+: >1%, nt. = not tested. ^(d)Gen. = genomic DNA, Org. = organelle DNAfrom chloroplasts (algae and plants) and mitochondria (fish).^(e)Amplicons as described in example 13.

1. A method of isolating genomic DNA from a sample containing intactcells, said method comprising (a) contacting said sample containingintact cells with a detergent and magnetic particles, said magneticparticles comprising an organic polymer, and whereby soluble genomic DNAfrom said sample is bound to the surface of said particles in asequence-independent manner in the presence of the detergent and absenceof any chaotropic agent, and (b) separating said magnetic particles withbound genomic DNA from the sample.
 2. The method as claimed in claim 1,wherein the detergent is anionic.
 3. The method as claimed in claim 2,wherein the detergent is sodium dodecyl sulphate, or another alkalimetal alkylsulphate salt, or sarkosyl.
 4. The method as claimed in claim1, wherein the concentration of detergent is 0.2 to 30% (w/v).
 5. Themethod as claimed in claim 1, wherein the detergent is contained in acomposition additionally comprising one or more monovalent cations,chelating agents or reducing agents.
 6. The method as claimed in claim1, wherein the detergent is used in alkaline solution.
 7. The method asclaimed in claim 1, wherein the magnetic particle has a negativelycharged, neutral or hydrophobic surface.
 8. The method as claimed inclaim 1, wherein the genomic DNA is eluted from the support, followingseparation from the sample.
 9. The method as claimed in claim 8, whereinthe genomic DNA is eluted by heating.
 10. The method as claimed in claim1, wherein the organic polymer is polyurethane.
 11. The method asclaimed in claim 1, wherein the organic polymer is polystyrene.
 12. Themethod as claimed in claim 1, wherein the organic polymer is latex. 13.The method as claimed in claim 1, wherein the magnetic particlecomprises superparamagnetic polystyrene beads.
 14. The method as claimedin claim 1, wherein the magnetic particle is porous.
 15. The method asclaimed in claim 1, the method further comprising the step of detecting,hybridizing, amplifying or quantifying the bound genomic DNA after theseparating step.
 16. A kit for isolating genomic DNA from a sample, thekit comprising (a) magnetic particles as defined in claim 1; (b) one ormore detergents; and (c) instructions for isolating genomic DNAaccording to the method of claim
 1. 17. A method of isolating RNA andgenomic DNA from a sample, said method comprising (a) isolating solublegenomic DNA from said sample using magnetic particles according to themethod of claim 1; (b) isolating RNA from said sample.
 18. A kit forisolating RNA and genomic DNA from a sample, the kit comprising (a)magnetic particles as defined in claim 17; (b) oligo dT beads; (c) oneor more detergents; and (d) instructions for isolating RNA and genomicDNA according to the method of claim
 17. 19. The kit for isolating RNAand genomic DNA from a sample according to claim 18, wherein themagnetic particle is a superparamagnetic polystyrene bead.
 20. A methodof isolating genomic DNA from cells in a sample, said method comprising(a) obtaining cells from said sample by immunomagnetic separation; (b)producing a lysate by contacting said cells with a detergent andmagnetic particles in the absence of any chaotropic agent, the magneticparticles comprising an organic polymer whereby soluble genomic DNA insaid lysate is bound to the surface of the support in asequence-independent manner in the presence of the detergent and absenceof any chaotropic agent; and (c) separating said magnetic particles withbound genomic DNA from said lysate.
 21. The method as claimed in claim20, wherein said cells are in a cell:bead complex.
 22. A method ofisolating RNA and genomic DNA from cells in a sample, said methodcomprising (a) obtaining cells from said sample by immunomagneticseparation; (b) producing a lysate by contacting said cells with adetergent and magnetic particles in the absence of any chaotropic agent,the magnetic particles comprising an organic polymer, and wherebysoluble genomic DNA in said lysate is bound to the surface of thesupport in a sequence-independent manner in the presence of thedetergent and absence of any chaotropic agent; (c) separating saidmagnetic particles with bound genomic DNA from said lysate; and (d)isolating RNA from said lysate.
 23. The method as claimed in claim 22,wherein said cells are in a cell:bead complex.
 24. A method of isolatinggenomic DNA from a sample, said method comprising (a) contacting saidsample with a detergent and magnetic particles comprising an organicpolymer wherein the surface of the magnetic particle has a negative,neutral or hydrophobic surface, and whereby soluble genomic DNA in saidsample is bound to the surface of said particles in asequence-independent manner; and (b) separating said magnetic particleswith the bound genomic DNA from the sample.